The present invention relates to a polypeptide, capable of being substituted for a Toxoplasma gondii antigen, a polynucleotide whose expression corresponds to said polypeptide, and their use for diagnostic and therapeutic purposes.
Toxoplasmosis is an infectious disease caused by a Toxoplasma gondii protozoal parasite, a member of the class Sporozoa, and of the order Coccidia. Toxoplasma gondii is an intracellular parasite which reproduces in a wide variety of cell types in its hosts, which are mammals.
This parasite, which is very widespread geographically, is an important pathogenic agent, not only in human medicine, but also in veterinary medicine.
In man, two forms of the parasite have been described: the xe2x80x9ctachyzoitexe2x80x9d, which is the multiplicative form encountered during the acute phase of the disease and the xe2x80x9cbradyzoitexe2x80x9d, a resistant form which persists encysted in the nervous tissues, and which is probably responsible for maintaining a durable immunity to reinfection.
In humans, toxoplasmosis is most often asymptomatic and most often passes unnoticed without any consequences. There are however cases for which a Toxoplasma infection or a reactivation of a previously acquired infection can generate serious disorders for the so-called at risk individuals who are pregnant women and immunodepressed and immunosuppressed subjects. This organism has multiple replication sites. Thus, it may be responsible for severe cerebral and ocular impairments when its replication site is the cells of the central nervous system and the cells of the reticuloendothelial system. Pregnant women represent high-risk subjects, since a Toxoplasma infection, especially during the first few months of pregnancy, may be responsible for serious fetal and neonatal complications if maternal treatment is not undertaken early and pursued assiduously. In particular, newborns contaminated via the transplacental route are subject to serious ocular and cerebral disorders which are even fatal in certain cases. Immunodepressed patients and particularly AIDS patients are subject to serious Toxoplasmosis due most often to reactivations of previous infections, although dissemination following a primary infection cannot be completely excluded (see FARKASH et al., J. Neurology, Neurosurgery and Psychiatry 1986, 49, 744-748 and Luft et al., J. Am. Med. Ass. 1984, 252, 913-917).
It is therefore essential to have available diagnostic tests which make it possible to determine the presence of the parasite, especially in pregnant women, either by detecting specific antibodies or by detecting Toxoplasma antigens in the subject.
HUGUES in xe2x80x9cCurrent topics in Microbiology and Immunologyxe2x80x9d (Vol. 120, 1985, SPRINGER Ed., pages 105-139) has listed a number of commercially available serodiagnostic tests such as the SABIN and FELDMAN staining test, standardized by BEVERLY and BEATTLE in 1958 and perfected by FELDMAN and LAMB (1966), WALDELAND (1976) and BALFOUR et al. (1982); the REMINGTON (1968) test for the detection of antibodies by immunofluorescence, optimized in 1975 by KARIM and LUDLAM; the hemagglutination tests; the ELISA test for the detection of antibodies specific for Toxoplasma, by the isolation of IgM in situ on a microplate described in 1983 by WIELARRD et al.
The different tests used are based on the formation of immune complexes between the Toxoplasma gondii antigens and their specific antibodies. One of the critical points therefore consists in the characterization of the Toxoplasma gondii major antigens, which induce a specific immune response and are capable of being used in serological detection tests.
Some authors have shown that most of the monoclonal antibodies directed against the Toxoplasma gondii surface recognize a protein of 27 to 35 KDa, called P30 (Kasper et al.; 1983, J. Immunol. 130, 2407-2412). Several studies have demonstrated that this P30 protein is a major surface antigen which can be used for the production of vaccines or in diagnostic tests, especially in immunoassays. Moreover, Boothroyd et al. (see Patent Application WO 89/08700) have identified and obtained the genetic material encoding the Toxoplasma gondii P30 protein and suggested the use of the gene for the production of recombinant protein, peptides and antibodies. This gene has been cloned (Burg et al., 1988, J. Immunol., 141, 3584-3591). Analysis of the sequence shows a potential N-glycosylation site, a secretory signal positioned at the N-terminal end which is cleaved in the mature P30 protein and a highly hydrophobic C-terminal region which is also cleaved and replaced by a glycolipid which allows membrane anchorage of the P30 protein.
There remains, however, a problem which consists in producing sufficient quantities of the P30 antigen. Indeed, three approaches are currently available, with the aim of obtaining antigens and parts of P30 antigens. which can be used especially in diagnostic procedures.
The first consists in performing cultures of the Toxoplasma gondii parasite in a large number of infected mice, in collecting the natural Toxoplasma gondii antigens, and especially P30, via the peritoneal route and in purifying them. However, this technique has many disadvantages such as the cost, the need of having available experienced staff and a large number of animals, the complexity of the technique or the difficulties linked with the extraction which is not very efficient.
The second approach, derived from the preceding one, involves the in vitro culture of the parasite in cell cultures (Grimwood et al. 1979, Experimental Parasitology, 48, 282-286). However, this approach does not make it possible to overcome the disadvantages of cost or extraction mentioned above.
The third approach refers to genetic recombination technologies and consists in introducing and cloning into a prokaryotic, eukaryotic or viral genome all or part of the gene encoding the P30 protein, in expressing said gene in a host cell and in extracting the recombinant P30 protein obtained. This technique offers many advantages but suffers from a low level of expression of the protein. Moreover, Burg et al. (1988, Journal of Immunology, 141, 3584-3591) has shown that the natural P30 protein is a highly structured antigen exhibiting conformational constraints. The conformation of the P30 protein is a critical element for its recognition by specific antibodies and results especially from the presence of disulphide bridges in the protein. Now, it is difficult, through genetic recombination, to perfectly control the post-translational modifications to which the synthesized proteins are subjected. Consequently, it is difficult to obtain, by genetic recombination, a P30 protein or a part of said protein which is appropriately matured so that it adopts a conformation which is sufficiently close to that of the natural P30 protein to allow specific recognition by antibodies directed against said protein.
In accordance with the document WO-A-89/12683, polypeptides are described which are capable of reacting with. an anti-Toxoplasma gondii P30 protein antibody and which possess a peptide sequence chosen from the sequence SEQ ID No.15, fragments of SEQ ID No.15, SEQ ID No.16, SEQ ID No.17 and SEQ ID No.18, which are identified at the end of the description. These polypeptides were determined from the peptide sequence of a Toxoplasma gondii protein, and consist of the sequence of the latter or of the antigenic fragments of the latter. Given the low level of homology of said sequences with that of the P30 protein, it can be stated that the peptide sequence of the protein at the origin of these polypeptides is not that of the P30 protein. These polypeptides pose the problem of their specificity; indeed, they have antigenic properties toward anti-P30 antibodies, but they probably have the same properties toward antibodies directed against the protein from which they were defined.
Surprisingly, it has been shown that a polypeptide synthesized by the chemical route or by genetic recombination methods, which has been subjected to no modification after its synthesis, is capable of being recognized by antibodies specific for the P30 protein, designated anti-P30 antibodies.
Before embarking on the subjects of the invention, various terms used in the description which will follow are defined below.
xe2x80x9cProtein P30xe2x80x9d is understood to mean the Toxoplasma gondii major antigen, therefore the peptide sequence is identified by the reference SEQ ID No.1.
xe2x80x9cSera from individuals or animals infected by Toxoplasma gondiixe2x80x9d is understood to mean especially sera from patients who have contracted a recent or previous Toxoplasma gondii infection and who contain immunoglobulins recognizing specifically all or part of the P30 protein.
Of course, a polypeptide according to the invention may also be recognized by other antibodies, such as for example monoclonal or polyclonal antibodies obtained by immunizing a variety of species with the natural P30 protein, the recombinant P30 protein or fragments or peptides thereof.
xe2x80x9cPolypeptidexe2x80x9d designates a peptide or a peptide fragment, or a protein, obtained by chemical synthesis or by genetic recombination techniques. The polypeptides according to the invention can be obtained by conventional methods of synthesis, for example in an automated peptide synthesizer, or by genetic engineering techniques comprising the insertion of a DNA sequence encoding said polypeptide into an expression vector such as a plasmid or a virus as described in the present application, and the transformation of cells with this expression vector and the culture of these eukaryotic or prokaryotic cells.
The degree of homology of a first peptide sequence, compared with a second reference peptide sequence, characterizes the maximum degree, expressed as a percentage, of identity between the two sequences. It is evaluated, for a succession of a given number of contiguous amino acids, by the alignment of the two sequences, and by displacing one relative to the other, and by comparing the amino acids in the two sequences.
xe2x80x9cSequence derived from another amino acid sequencexe2x80x9d is understood to mean an amino acid sequence modified by insertion and/or deletion and/or substitution and/or extension and/or shortening and/or chemical modification of one or more amino acids, excluding any peptide sequence in which any succession of 20 contiguous amino acids exhibits at least 40% homology with the peptide sequence of the P30 protein (SEQ ID No.1), so long as these modifications preserve substantially or even enhance the properties of said sequence. Thus, xe2x80x9cderived sequencexe2x80x9d is understood to mean especially the sequences in which one or more amino acid(s) is (are) substituted by one or several other amino acid(s) as illustrated in Example 6; the sequences in which one or more amino acid(s) of the L series is (are) replaced by an amino acid of the D series, and vice versa; the sequences into which there has been introduced a modification of the side chains of the amino acids, such as for example an acetylation of the amine functional groups, a carboxylation of the thiol functional groups, an esterification of the carboxylic functional groups; a modification of the peptide bonds such as, for example, of the carba, retro, inverso, retro-inverso, reduced and methyleneoxy bonds.
xe2x80x9cPolynucleotidexe2x80x9d is understood to mean either a DNA sequence, or an RNA sequence, or a cDNA sequence resulting from the reverse transcription of an RNA sequence, of natural or synthetic origin, with or without modified
The subject of the present invention is a polypeptide capable of reacting specifically with an anti-Toxoplasma gondii P30 protein antibody, and comprising a peptide sequence, having, in any succession of 6 contiguous amino acids, at most 67% homology with the peptide sequence of said P30 protein, as identified by SEQ ID No.1, or comprising a sequence derived from said peptide sequence.
Preferably, a polypeptide of the invention comprises a peptide sequence, having, in any succession of 11 contiguous amino acids, at most 36% homology with said peptide sequence SEQ ID No.1, or comprises a derived sequence. A specific polypeptide of the invention possesses a peptide sequence consisting of a peptide sequence, having, in any succession of 11 contiguous amino acids, at most 36% homology with said peptide sequence SEQ ID No.1, or consisting of a derived sequence.
The subject of the present invention is also a polypeptide capable of reacting specifically with an anti-Toxoplasma gondii P30 protein antibody, which possesses a peptide sequence comprising SEQ ID No.2, or a derived sequence.
The peptide sequence of such a polypeptide advantageously comprises a sequence chosen from SEQ ID No.3, SEQ ID No.4, and their respective derived sequences.
In particular, said peptide sequence comprises a sequence chosen from SEQ ID No.5, SEQ ID No.6, and their respective derived sequences.
Still more preferably, said peptide sequence comprises a sequence chosen from SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, and their respective derived sequences.
A specific polypeptide of the invention possesses a peptide sequence consisting of a sequence chosen from SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, and their respective derived sequences.
The polypeptide as defined above may be of phage or viral or bacterial or lower eukaryotic or higher eukaryotic origin.
In particular, this polypeptide may be an envelope peptide fragment, a membrane peptide fragment or a peptide fragment capable of being excreted.
Moreover, said polypeptides may be conjugated, according to techniques well known to persons skilled in the art, to a carrier molecule such as for example a natural or recombinant protein (with the exception of the natural or recombinant P30 protein), a synthetic polymer of amino acids or of aliphatic chains, a nucleic fragment or to a tracer molecule such as for example an oligonucleotide, an enzyme such as especially horseradish peroxidase, alkaline phosphatase or galactosidase, or alternatively a radioelement or attached to any support.
It is clearly understood that any peptide sequence different from those described in the invention, and likely to be contained in the peptide sequence of a polypeptide of the invention should not, as such, be capable of reacting with an anti-P30 protein antibody.
A second subject of the present invention is an application of a polypeptide as described above and consists of a reagent for the detection of a Toxoplasma gondii infection, said reagent comprising, as reactive substance, a polypeptide of the invention.
A third subject of the invention is a kit for the detection of a Toxoplasma gondii infection, comprising the reagent described above, supported by a solid support, immunologically compatible with said reagent.
The term solid support as used here is, without limitation, in the form of a microtiter plate, a sheet, a cone, a well, a bead, or any other appropriate micro-particulate substrate, and includes all materials on which peptide fragments of the invention can be immobilized. This may be synthetic materials which are chemically modified or otherwise, especially polysaccharides, such as cellulose materials, for example paper, cellulose derivatives such as nitrocellulose and cellulose acetate; polymers such as vinyl chloride, polyethylene, polystyrene, polyacrylate, or copolymers such as propylene and vinyl chloride polymer, vinyl chloride and vinyl acetate polymer; styrene-based copolymers; natural fibers such as cotton and synthetic fibers such as nylon. Preferably, the solid support is a polystyrene polymer, a butadiene/styrene copolymer or a butadiene/styrene copolymer mixed with one or more polymers or copolymers chosen from polystyrene, styrene/acrylonitrile or styrene/methyl methacrylate copolymers, polypropylenes, polycarbonates and the like.
The attachment of the reagent to the solid support may be performed in a direct or indirect manner.
In a direct manner, two approaches are possible: either by adsorption of the reagent onto the solid support, that is to say via non-covalent bonds (mainly of the hydrogen, Van der Waals or ionic type), or by establishment of covalent bonds between the reagent and the support. In an indirect manner, an xe2x80x9canti-reagentxe2x80x9d compound capable of interacting with the reagent can be attached beforehand (by adsorption or covalent bonding) onto the solid support so as to immobilize the whole on the solid support.
The invention provides, in addition, a process for the detection and/or separation, and especially purification, and/or assay of anti-P30 protein antibodies in a sample of biological fluid comprising the following steps: a said sample is brought into contact with the reagent of the invention, under conditions which allow an immunological reaction, and then the immune complex which may be formed is detected, separated and/or quantified.
According to a variant, the invention provides a process for assaying the Toxoplasma gondii P30 protein in a sample of a biological fluid, which is carried out by a competition technique, during which said sample is brought into contact simultaneously with a predetermined quantity of anti-P30 protein antibodies, and a predetermined quantity of a reagent of the invention, and the quantity of P30 protein in said sample is determined by deduction from the measured quantity of the complex formed between the reagent and said anti-P30 protein antibodies.
According to another variant of a process of the invention for assaying the P30 protein in a sample of a biological fluid, in a first instance, said sample is brought into contact with a predetermined quantity of anti-P30 protein antibodies, in a second instance, a predetermined quantity of a reagent of the invention is added and the quantity of P30 protein in said sample is determined by deduction from the measured quantity of the complex formed between the reagent and said anti-P30 protein antibodies.
These processes may be based on a radioimmunological method of the RIA, RIPA or IRMA type or an immunoenzymatic method of the Western-blot or ELISA type.
Another application of the polypeptide of the invention is an active immunotherapeutic composition, especially a vaccinal preparation, comprising, as active ingredient, a polypeptide described above, said active ingredient being optionally conjugated with an immunologically appropriate support, and optionally a pharmaceutically acceptable excipient.
The invention provides, in addition, a polynucleotide encoding a polypeptide described above. This polynucleotide comprises a nucleotide sequence especially chosen from SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQ ID No.14.
It also relates to a functional expression cassette allowing the expression of a polynucleotide of the invention and comprising the latter, as well as a vector and a eukaryotic or prokaryotic cellular system comprising an expression cassette.